Field of the Invention
The present invention relates to a composition including plant-derived recombinant human serum albumin, lipids, and plant protein hydrolysates as active ingredients for cryopreservation of stem cells or primary cells, and more particularly, to a composition including plant-derived recombinant human serum albumin, lipids, and plant protein hydrolysates as active ingredients for cryopreservation of stem cells or primary cells, in which the albumin, lipids, and plant protein hydrolysates are present in sufficient quantities to reduce cell death caused by freezing while maintaining animal-free and xeno-free conditions during long-term preservation of stem cells or primary cells; and to a method for cryopreservation of stem cells or primary cells using the composition.
Description of the Related Art
Stem cells refer to cells that can proliferate indefinitely in an undifferentiated state as well as differentiating to have a specialized function and shape when specific environments and conditions are provided. Since human embryonic stem cells are capable of continuous self-renewal and pluripotency like differentiating into all cell types of the human body, under appropriate in vitro culture conditions, the range of applications of studies on human embryonic stem cells are expanding to a wide variety of fields including basic studies for understanding development, differentiation, and growth of the human body, development of cell therapy products for the treatment of damages or various diseases of the human body, and efficacy screening for various candidate therapeutic drugs, disease etiology study, development of therapy strategies, etc. Even though demands for adult stem cells and human embryonic stem cells are rapidly increasing, there is an obstacle to development of the related technology because of a lack of techniques and methods for maintenance and long-term preservation of undifferentiated adult stem cells and human embryonic stem cells.
Further, primary cells refer to normal cells that are obtained by a direct primary culture of animal tissues or organs, directly obtained from living organisms. When these cells are cultured, they grow and go through cell divisions to a certain extent, but have a limitation as they undergo senescence after a certain number of passages. Despite such limitation, primary cells are still used in production of biomedical products due to an advantage of showing responses similar to those of a living organism. However, cryopreservation of cells is required because of the limitation.
Therefore, to use stem cells or primary cells in a clinical test, cell therapy, etc., it is very important to develop a preservation solution containing no animal-derived factors and a preservation method suitable for primary cells and stem cells, respectively.
Cold preservation and cryopreservation are representative methods for a method of preserving stem cells while maintaining their survival rate and characteristics such as pluripotency or multipotency. The cold preservation is a method of preserving stem cells at a low temperature, which was developed for the purpose of short-term transportation and storage. However, cold preservation method is suitable only for short-term preservation for 1 week or shorter, more accurately, 1 to 3 days at a low temperature (4° C.), and thus there is a limitation in use of cold preservation for long-term preservation of stem cells and primary cells. In contrast, cryopreservation is a method of preserving cells, tissues, and organs at an ultra-low temperature of −70° C. to −196° C., at which all metabolic processes in cells come to a halt, thereby semi-permanently preserving desired stem cells. This method is the most ideal method for semi-permanent preservation of stem cells and primary cells for research and therapeutic purposes, and is used for preservation of stem cells and primary cells in all nonprofit or commercial cell banks.
However, it is difficult to avoid cell damage caused by freezing and thawing processes which are inevitably accompanied by ice crystal formation and ionic and osmotic imbalance. Thus, there is increasing concern about the damage due to freezing and thawing processes. In order to minimize the damage and maximize survival rate and stemness after freezing and thawing processes, it is very important to develop cryopreservatives and cryopreservation methods according to biochemical and physical characteristics of corresponding cells for minimizing damages caused by freezing and thawing processes.
In general, intracellular ice crystallization which occurs during cryopreservation of animal cells is the main cause of cell damage. In order to prevent it, a cryoprotectant is used, and dimethyl sulfoxide (DMSO) or glycerol is mainly used. They are permeating-type cryoprotectants which effectively reduce ice crystallization during freezing and thawing processes. In detail, glycerol functions as a salt-buffer to bind to metal ions and to dehydrate cells themselves, thus inhibiting cell damage by reducing the volume of growing ice crystals during freezing of intracellular water. DMSO partially dissolves the cell membrane to prevent formation of a puncture and interferes with hydrogen bonding between water molecules to inhibit cell damage caused by ice crystals. However, there are still problems that cell damage is inevitable because DMSO and glycerol are cytotoxic, ionic and osmotic imbalance occurs, and active oxygen free radicals are generated during freezing and thawing processes.
Accordingly, in order to solve the problems to some extent, a method of combining cryoprotectants with a vehicle capable of minimizing ionic and osmotic imbalance and generation of active oxygen free radicals has been widely used. However, fetal bovine serum (FBS) or human serum which is generally used as a component of a cryopreservation liquid is also problematic in that their quality control is difficult, because of lot to lot variation, and that human-derived primary cells and stem cells to be stored can be exposed to infectious agents transmitted from animals (prion, virus, etc., which serves as the cause of bovine spongiform encephalopathy) or human serum, and may cause immune responses. To eliminate the problems of quality control of the cryopreservation liquid components and infectious agents, a technology using a recombinant human serum albumin produced from microorganisms or animal cells has been recently reported. However, the use of the recombinant human serum albumin also partially has the problem of quality control of cryopreservation liquid components, and risks of exposure to infectious agents which can infect animals still remains. Therefore, there is a need for development of new cryopreservation liquids.
Further, since kinds of components included in a cryopreservative composition and a composition ratio thereof may vary depending on types of cells to be cryopreserved, it is required to investigate components suitable for stem cells and primary cells and a proper composition ratio thereof for development of a composition for effective cryopreservation of stem cells and primary cells. In order to develop a composition for cryopreservation of stem cells or primary cells with both safety and stability, it is required to develop xeno-free and animal-free components which are effective for cryopreservation of stem cells and primary cells and a proper composition ratio thereof.